Model systems which mimic receptor binding and other cell-based assays are of increasing importance in molecular biology. Phenomena such as transporter activity, pre- and post-synaptic cell interactions, chemotactic response, enzyme-ligand binding and the like are of relevance to pharmacology, clinical chemistry and basic research.
For example, one clinically relevant system involves the interaction of cells at synapses. At least two general types of synapses exist in nature. In electrical synapses, gap junctions connect cells which are in communication. Gap junctions permit direct transmission of electrical impulses from a presynaptic cell to a postsynaptic cell.
In the more common chemical synapse, an axon terminal of a presynaptic cell contains vesicles filled with a neurotransmitter, such as epinephrine or acetylcholine, which is released by exocytosis when a nerve impulse reaches the axon terminal. The vesicles release their contents into the synaptic cleft and the transmitter diffuses across the synaptic cleft. After a brief lag time (e.g., about 0.5 ms) the transmitter binds to receptors on postsynaptic cells. This typically causes a change in ion permeability and electrical potential in the postsynaptic cell. Excitatory signals induce an action potential in the postsynaptic neuron. Inhibitory signals prevent production of an action potential in the postsynaptic neuron. Both inhibitory and excitatory signals can exist simultaneously in the same synaptic cleft, depending on the cell types, neurotransmitters, etc. Similarly, a postsynaptic cell can be in simultaneous contact with multiple presynaptic cells, each of which can transmit both excitatory and inhibitory signals to the postsynaptic cell.
The presence of transmitter in the synapse is regulated in a variety of ways, thereby controlling the signal received by the postsynaptic cell. For example, some cells and vesicles actively transport transmitter out of the synapse, thereby reducing the presence of the transmitter in the junction. Similarly, oxidases and other enzymes degrade some neurotransmitters in the synapse. Neurotransmitters can also diffuse away from the synapse. For a review of neurotransmitter and transporter systems, see, Neurotransmitter Transporters: Structure, Function and Regulation (1997) M. E. A. Reith, ed. Human Press, Towata N.J., and the references cited therein.
Transporters have a variety of important biological roles. For example, the Na+/Clxe2x88x92 dependent transporters (e.g., the monoamine transporters, as well as betaine, creatine, GABA, glycine, proline and taurine carriers) are the primary sites of action for a variety of drugs of both therapeutic and abuse potential. For example, among the monoamine transporters, inhibition of the dopamine transporter (DAT) is linked to euphoric and reinforcing properties of psychomotor stimulants such as cocaine and amphetamines. The major classes of therapeutic antidepressants act by inhibiting the norepinephrine and serotonin transporters (NET and SERT) and many of these compounds have proved clinically useful in the treatment of panic, stress, obsessive compulsive disorders, and other conditions.
Chemotactic responses have also long been known to play significant roles in various biologicial systems. Chemotaxis is the capacity of a motile cell to respond to chemical changes in its environment by directed movement. The migration of a motile cell exhibiting a chemotactic response can be either up or down a concentration gradient of a chemotactic factor. For example, phagocytic cells like macrophages are attracted by and move toward various substances generated in an immune response, whereas other motile cells including certain bacteria can move either toward an attractant (e.g., assorted sugars) or away from various repellents (e.g., phenol). For further discussion of chemotaxis and related components, including adhesion and chemotactic factors, see, Kuby, Immunology, 3rd Ed. W. H. Freeman and Company, New York (1997) and Stryer, Biochemistry, 4th Ed., W. H. Freeman and Company, New York (1995).
Aside from methods and devices for modeling transporter activity and chemotactic responses, general binding assays for studying, e.g., enzyme-ligand binding interactions, receptor-ligand binding interactions, and the like are also useful, e.g., in modeling biological systems.
In general, existing in vitro systems for studying transmitters, transporters, presynaptic and postsynaptic cells, and other aspects of cell signaling do not provide ideal high-throughput methods and devices for modeling and mimicking transmitter diffusion, transporter activity, transmitter activity, and the like. More generally, cell-cell signaling, which is central to biological activity, is not ideally modeled using existing technologies and additional high throughput methods of screening for modulators of signaling activities are desirable. Furthermore, progress in the study of chemotaxis and various binding activities has also been impeded by in vitro assays that are tedious to perform and whose results have been difficult to quantify. As such, automated and quantitative assays for all of these important biological processes are desirable.
The present invention provides these and other features by providing high-throughput microscale systems for modeling transporter activity, transmitter degradation activity, transmitter activity, cell signaling, and detection of modulators (inhibitors and enhancers) of transporter or transmitter degradation activity. The present invention also relates to high-throughput systems for modeling gradient induced activities, e.g., chemotactic responses, and for assessing general binding activities. These and many other features which will be apparent upon complete review of the following disclosure.
The invention provides methods, devices, kits, reagents and related materials for modeling various important biological processes. For example, the present invention is optionally used to determine the activity of transporter components such as neurotransmitter transporters (for example, the neurotransmitter acetylcholine is specifically internalized by cells via endocytosis of acetylcholine from the synaptic cleft during the recovery period following signal transmission). The invention is also optionally used to assess gradient induced activities (e.g., study chemotactic responses) and to evaluate the binding activity of, e.g., various biological components. The methods are typically conducted in a microscale format using a microfluidic system which includes or is coupled to sources of the relevant assay components.
In the transporter-related methods and assays of the invention, a first component which includes transporter activity is flowed through a first channel. A second component which produces a detectable signal upon exposure to a transportable molecule or set of transportable molecules is flowed into the first channel. The transportable molecule is flowed into the first channel and a signal produced by contacting the second component with the transportable molecule is then detected. Typically, the level of signal product is inversely related to transporter activity.
A variety of formats for the methods are appropriate. The first and second components are typically flowed sequentially (a typical configuration) or simultaneously in the first channel. The second component optionally is flowed into contact with the transportable molecule in the presence or absence of the first component (for example, if flowed in the absence of the first component, the resulting signal serves as a positive control for the signal produced by contacting the second component with the transportable molecule).
Known activity modulators are optionally incorporated into assay schemes as controls for modulation of a particular transporter. For example, paraxetine, citalopram, fluxetine, imipramine, amitriptyline, mazindol, cocaine, desipramine, nomifensine, GBR12909, D-amphetamine, L-amphetamine, nortriptyline, DA, MPP+, NE, and 5-HT are known inhibitors of the transport of human monoamine clones. See, Neurotransmitter Transporters: Structure, Function and Regulation, e.g., at chapter 1 (1997) M. E. A. Reith, ed. Human Press, Towata N.J., and the references cited therein.
The first component is typically a cell or component with similarity to a cell such as a cell membrane (or other lipid membrane preparation) having transporter activity. Similarly, the second component is typically, e.g., a cell, cell membrane (or other lipid membrane preparation), or other biological or synthetic moiety comprising a receptor for the transportable molecule which is capable of producing a detectable signal upon exposure to a transportable molecule (e.g., a transmitter). The first or second components typically include a transporter or transmitter receptor carrier moiety or set of carrier moieties which includes a receptor or transporter. A xe2x80x9ccarrierxe2x80x9d is a component comprising the specified activity (e.g., transporter, transmitter, transmitter receptor, etc.). Examples of carriers or carrier sets include cells, liposomes, organelles, proteins, and protein-lipid complexes. Examples of transportable molecules or set of transportable molecules include proteins, sets of proteins, peptides, sets of peptides, lipids, sets of lipids, carbohydrates, sets of carbohydrates, organic molecules, sets of organic molecules, drugs, sets of drugs, receptor ligands, sets of receptor ligands, antibodies, sets of antibodies, neurotransmitters, sets of neurotransmitters, cytokines, sets of cytokines, chemokines, sets of chemokines, hormones, sets of hormones and a variety of other biologically active and inactive molecules. In preferred aspects, the first component is a carrier moiety or set of carrier moieties comprising a transporter activity having neurotransporter activity.
Preferred transporter components include cells which specifically or non-specifically internalize transmitter molecules, e.g., by specific or non-specific endocytosis, or pinocytosis. These transporter molecules, such as the Na+/Clxe2x88x92 dependent transporters (e.g., the monoamine transporters, as well as betaine, creatine, GABA, glycine, proline and taurine carriers), transport corresponding transportable molecules such as acetylcholine, catecholamines (e.g., epinephrine, norepinephrine, dopamine, serotonin, and other adrenergic neurotransmitters), endorphins (e.g., xcex1 and xcex2-endorphin), enkephalins (e.g., Met-enkephalin or Leu-enkephalin), somatostatin, leutinizing hormone-releasing hormone, thyrotropin-releasing hormone, substance P, angiotensin I, angiotensin II, vasoactive intestinal peptide, serotonin, and gamma-aminobutyric acid (GABA).
In one aspect of the invention, the transportable molecule is flowed from a second channel into the first channel and the second component is flowed from a third channel into the first channel, where the first component, the second component and the transportable molecule mix. For example, the second and third channels optionally intersect the first channel in a mixing region, where the first, second and third components diffuse into contact in the mixing region. An advantage to this arrangement is that the diffusion mimics diffusion of components in synapses in vivo, providing a convenient way of modeling transport of biologically active transportable molecules such as neurotransmitters. The first and second components are optionally flowed sequentially, serially or concomitantly. Potential transport modulatory compounds (e.g., inhibitors) are optionally flowed into contact with the first component to test for an effect on transport of the transportable molecule. For example, the modulatory compound is optionally flowed into the first channel prior to introduction of the second component and the transportable molecule, or concomitant with flow of the transportable molecule, depending on the format of the particular assay.
In one embodiment, concentration of the transportable molecule is decreased in solution in the first channel as the first component internalizes the transportable molecule. In other aspects, the first component sequesters or otherwise inactivates the transportable molecule.
A detectable signal produced by transport of the transportable molecule, or inhibition of transport of the transportable molecule provides an indication of, e.g., the transporter activity present in the first component, or an ability of the inhibitor to inhibit the transporter activity present in the first component, or an ability of the second component to sequester the transportable molecule. For example, the detectable signal is optionally a cellular activity, a light emission, a radioactive emission, a change in pH, a change in temperature, or the like. The concentration of the first component, the transportable molecule, or the second component (or any combination of these components), as well as potential modulators is optionally varied in the first channel and the resulting increase or decrease in signal strength is typically measured.
The present invention also relates to methods of detecting a gradient induced activity. The methods include providing a first channel, e.g., a microchannel, with an internal surface including a first and a second longitudinal segment. A first component (e.g., an adhesion factor) or a set of first components is typically attached to a region of the first longitudinal segment and a second component, such as a motile cell (e.g., a phagocytic, a protozoic, a moneran cell, etc.) is generally attached to the first component or to one or more members of the set of first components. The second component is, e.g., optionally fluorescently labeled. A gradient is typically formed from an edge of the second longitudinal segment of the first channel, which induces the second component to detach from the first component or from the one or more members of the set of first components. Thereafter, a detectable signal produced by the detached second component is generally detected. The type of gradient utilized with these methods optionally include a chemical composition gradient, a light energy gradient, a magnetic gradient, a pH gradient, a dissolved oxygen gradient, a temperature gradient, or the like.
The first component or set of first components are optionally attached to the first channel by flowing the first component or the set of first components over the first longitudinal segment of the first channel concomitantly with flowing a third component (e.g., a buffer) over the length of the second longitudinal segment of the first channel. During this step, at least some of the first component or set of first components attaches to the first longitudinal segment. Thereafter, the third component is flowed through the first channel to remove any unattached first component. The second component is typically then flowed through the first channel and in so doing, some of the second component attaches to the attached first component. This step is generally followed by flowing the third component through the first channel to remove any unattached second component.
In one preferred embodiment, the chemical composition gradient is established by concomitantly flowing a third component (e.g., a buffer) and a fourth component (e.g., a chemotactic factor) or a set of fourth components into the first channel in which the fourth component or the set of fourth components forms the gradient from an edge of the second longitudinal segment of the first channel. For example, a buffer is typically flowed from a second channel into the first channel and a chemotactic factor is typically flowed from a third channel into the first channel in which the buffer and the chemotactic factor mix in the first channel to form the gradient of the chemotactic factor. In one embodiment, the concentration of the fourth component or the set of fourths components is highest along a length of the second longitudinal segment that is farthest from the first longitudinal segment and lowest along the length of the second longitudinal segment that is nearest to the first longitudinal segment. In another embodiment, the concentration of the fourth component or the set of fourths components is lowest along a length of the second longitudinal segment that is farthest from the first longitudinal segment and highest along the length of the second longitudinal segment that is nearest to the first longitudinal segment.
As a negative control, e.g., a buffer is optionally flowed into contact with the attached motile cell in the first channel to assess the signal produced in the absence of the chemotactic factor. Additionally, a positive control optionally includes flowing, e.g., a chemotactic factor into contact with the attached motile cell in the first channel to determine the signal produced in the absence of a buffer.
The various components of these methods (e.g., the first, second, third and the fourth or more components) are optionally flowed, e.g., using a fluid direction component including, e.g., a fluid pressure force modulator, an electrokinetic force modulator, a capillary force modulator, a fluid wicking element, and/or the like. The methods optionally further include flowing a modulator into contact with the second component in the first channel prior to introduction of the fourth component in which the modulator modulates (e.g., activates or inhibits) detachment of the second component from the first component or the set of first components.
The detectable signal provides an indication of the gradient induced activity present in the second component and/or an ability of the modulator to modulate the gradient induced activity of the second component. The detectable signal optionally includes a refractive index, a cellular activity, a light emission, an absorbance, a change in absorbance, a fluorescence, a change in fluorescence, a color shift, a fluorescence resonance energy transfer, a radioactive emission, a change in pH, a change in temperature, a change in mass (e.g., by mass spectroscopy), or the like. Additionally, the chemical composition gradient formed by the fourth component or the set of fourth components in the first channel is optionally varied and a resulting increase or decrease in the detectable signal is optionally measured. Furthermore, the concentration of the second component is optionally increased in solution in the first channel as the gradient induces the second component to detach from the first component or the set of first components.
The present invention also relates to methods of detecting a binding activity. For example, a first component is typically flowed (e.g., in a first flow stream) through a first channel (e.g., a microchannel) concomitantly with at least one second component (e.g., in a second flow stream) or a set of second components in which the second component (e.g., an enzyme or a receptor) or the set thereof binds to the first component. Thereafter, the methods include, e.g., detecting a detectable signal that indicates a final concentration of the at least one first component or the set of first components that remains unbound after exiting from the first channel. Optionally, the methods include detecting a detectable signal that indicates an initial concentration of the at least one first component or the set of first components prior to entry of the component or set thereof into a first channel.
The first component or set of first components can diffuse more rapidly in solution than the second component or set of second components. Furthermore, the first channel generally includes a mixing longitudinal segment in which, during the flowing step, the first component or the set of first components diffuse substantially across the first channel in the mixing longitudinal segment, while the second component or the set of second components typically diffuse less than substantially across the first channel in the mixing longitudinal segment. The first and second components are typically flowed through the first channel using fluid direction components that optionally include, e.g., a fluid pressure force modulator, an electrokinetic force modulator, a capillary force modulator, a fluid wicking element, or the like.
As a positive control for detecting a detectable signal, the first component or the set of first components is optionally flowed through the first channel, e.g., in the absence of the second component or the set of second components. A negative control for detecting a detectable signal optionally includes the step of flowing the second component or the set of second components through the first channel, e.g., in the absence of the first component or the set of first components.
The methods also optionally include concomitantly flowing a modulator into contact with the second component in the first channel, in which the modulator modulates (e.g., activates or inhibits) the binding of the second component to the first component. The detected binding activity provides an indication of the binding activity of the second component and/or an ability of the modulator to modulate the binding activity of the second component. Additionally, the first and second detectable signals optionally include, e.g., a refractive index, a cellular activity, a light emission, an absorbance, a change in absorbance, a fluorescence, a change in fluorescence, a color shift, a fluorescence resonance energy transfer, a radioactive emission, a change in pH, a change in temperature, a change in mass (e.g., by mass spectroscopy), or the like.
In one preferred embodiment, the invention provides methods of detecting neurotransporter activity in a cell. In the methods, a first cell or cell set which includes a transporter activity is flowed in a first microscale channel. A selected neurotransmitter and a second cell or second cell set comprising a receptor for the neurotransmitter are flowed into the first channel. A signal produced by contact of the second cell or cells of the second cell set by the neurotransmitter is then detected, thereby determining the rate of transport activity of the transporter in the first cell or cells of the first cell set.
Optionally, the first cell or cell set is flowed into contact with the neurotransmitter prior to contacting any remaining neurotransmitter to the second cell or cell set. A transport inhibitor is optionally added to the microchannel and the resulting modulation in signal intensity produced by the second cell or cell set is measured, thereby determining the activity of the inhibitor on transport activity in the first cell set.
The invention provides devices and systems for practicing the methods noted herein. For example, in one aspect, a device which includes a body structure having at least a first, second and third microscale channel fabricated therein is provided. The first microscale channel typically includes a first component comprising transporter activity which transports at least a first transportable molecule. The second microscale channel intersects the first microscale channel at a first channel intersection. The second microscale channel typically includes a transportable molecule. The third microscale channel intersects the first microscale channel in a second channel intersection region. The third microscale channel includes a second component which binds to the first transportable molecule, causing emission of a detectable signal.
The device optionally includes a source of a modulatory agent such as an inhibitor which inhibits transport of the first transportable molecule by the first component, or an activator which enhances transport of the first transportable molecule. Sources of the other assay components noted herein, such as carrier moieties or sets of carrier moieties (including cells, liposomes, organelles, proteins, protein-lipid complexes, etc.), transportable molecules (proteins, sets of proteins, peptides, sets of peptides, lipids, sets of lipids, carbohydrates, sets of carbohydrates, organic molecules, sets of organic molecules, drugs, sets of drugs, receptor ligands, sets of receptor ligands, antibodies, sets of antibodies, neurotransmitters, sets of neurotransmitters, cytokines, sets of cytokines, chemokines, sets of chemokines, hormones, sets of hormones, etc.) are also optionally incorporated into the devices herein.
Devices optionally incorporate additional elements such as detectors for detecting signals produced in the first channel, e.g., operably connected to a computer for data analysis, fluidic controllers for directing fluid movement in the first channel, one or more transparent detection window fluidly connected to the first channel, robotic armatures for moving the body structure or sample arrays. Systems and devices typically incorporate, or are used in conjunction with, a computer having an instruction set for controlling or processing a signal from the detector, the fluidic controller, the robotic armature or other device or system elements.
The first and second intersections are optionally opposed across the first channel, or at least in a close proximal relationship. This arrangement is advantageous for studying biological diffusion properties of transporters, transportable molecules and transport receptors across small distances, e.g., to examine diffusion properties at, e.g., neural junctions between nerve cells (e.g., axons and dendrites).
In one embodiment, during operation of the device, a mixture of the first component, the second component and the transportable molecule are flowed in the first channel and the device has a detector for detecting a signal produced by the mixture. The detector is typically positioned to detect a signal produced by the mixture at multiple points in the first channel.
The present invention also includes a device or system including a body structure that includes a first microscale channel fabricated therein. The first microscale channel includes a first component (e.g., an adhesion factor) or a set of first components that includes a first attachment activity, in which the first component or set thereof is attached to a region of a first longitudinal segment of the first microscale channel. The first microscale channel also includes a second component (e.g., a motile cell) that includes a second attachment activity in which the second component is attached to the first component or to one or more members of the set of first components. The first microscale channel also includes a third component (e.g., chemotactic factor) or a set of third components that forms a gradient from an edge of a second longitudinal segment of the channel, in which the gradient induces the second component to detach from the first component or the members of the set of first components to produce a detectable signal.
In one embodiment, the depth of the first longitudinal segment differs from the depth of the second longitudinal segment in the first microscale channel of the device. Furthermore, the second component (e.g., a phagocytic cell, a protozoic cell, a moneran cell, etc.) are optionally fluorescently-labeled. The first microscale channel also optionally includes a modulator (e.g., an activator or an inhibitor) that modulates detachment of the second component from the first component.
Additionally, the device or system typically includes a detector in or proximal to the first microscale channel for detecting a signal produced in the first microscale channel that is operably connected to a computer. The device also generally include a fluidic controller for directing fluid movement in the first microscale channel, one or more transparent detection windows fluidly connected to the first microscale channel, a robotic armature for moving the body structure or sample plates relative to the body structure, and/or a source of components (e.g., microwell plates). When the device includes a computer, the computer typically includes an instruction set for controlling or processing a signal from the detector, the fluidic controller, and/or the robotic armature.
During operation of the device, a mixture of the third component (e.g., a chemotactic factor) or, e.g., the set of the third components and a buffer are optionally flowed in the first microscale channel and the device typically further includes a detector for detecting a signal produced by detachment of the second component induced by the third component in the mixture. Alternatively, the device includes a detector for detecting a signal produced by detachment of the second component induced by the third component at multiple points in the first channel.
The present invention also relates to a device or system that includes a body structure including at least a first, second, and third microscale channel fabricated therein. The first microscale channel includes a first and second component or sets thereof in which the second component (e.g., an enzyme or a receptor) binds to the first component. The second microscale channel typically intersects the first microscale channel at a first channel intersection and optionally the second microscale channel includes a second detector in or proximal to the second microscale channel for detecting an initial concentration of the first component. The third microscale channel optionally intersects the first microscale channel at a second channel intersection in which the third microscale channel includes a first detector in or proximal to the third microscale channel for detecting a final concentration of the first component that remains unbound. The first microscale channel of the device also optionally includes a modulator (e.g., an inhibitor) that modulates the second component from binding to the first component.
The first component or set of first components generally diffuses more rapidly in solution than the second component or set of second components. Furthermore, the first channel typically includes a mixing longitudinal segment in which, during operation of the device, the first component or the set of first components diffuse substantially across the first channel in the mixing longitudinal segment, while the second component or the set of second components diffuse less than substantially across the first channel in the mixing longitudinal segment.
The device also optionally includes a fluidic controller for directing fluid movement in the first microscale channel, a transparent detection window fluidly connected to the first microscale channel, a robotic armature for moving the body structure, and/or a source of components. When the device includes a computer, the computer typically includes an instruction set for controlling or processing a signal from the detector, the fluidic controller, and/or the robotic armature.
The invention also provides kits for practicing the methods and utilizing the devices noted herein. For example, the kits of the invention optionally include a first component comprising a transporter activity and a second component which is capable of producing a signal upon exposure to a transportable molecule which is transportable by the first component. The components generally include a carrier moiety or set of carrier moieties, a container for packaging the first or second component, instructions for practicing the methods herein, e.g., using the devices noted herein, one or more reagents for buffering or storing the first or second component, one or more transportable molecule, one or more test compound, a test compound library or the like.
Definitions
Unless otherwise indicated, the following definitions supplement those in the art.
xe2x80x9cTransporter activityxe2x80x9d refers to movement of a transportable molecule such as a transmitter across a biological barrier such as a cell membrane. This is typically performed by specific transporters or non-specific endocytosis of the transmitter or other transportable molecule. See, e.g., Darnell et al. (1990) Molecular Cell Biology, Second Edition Scientific American Books New York, FIGS. 17-38 for an introduction to specific endocytosis of a neurotransmitter. Additional details regarding neurotransporters are found in Neurotransmitter Transporters: Structure, Function and Regulation (1997) M. E. A Reith, ed. Human Press, Towata N.J. An example class of transporters are the biogenic amine transporters (e.g., those which transport norepinephrine (NE), dopamine (DA), or serotonin (5-HT), referred to as NET, DAT and SERT), which utilize ionic gradients of Na+, K+ and Clxe2x88x92 ions to drive transport reactions.
In addition to standard neurotransmitter transporters, transporter activity as used herein optionally includes other systems for translocating transmitters across a cell or other membrane, such as translocation through ionophores or other membrane translocation proteins such as permeases which facilitate transport of materials by mechanisms other than endocytosis. See, e.g., Darnell et al. (1990) Molecular Cell Biology Second Edition Scientific American Books New York, Chapter 15 for an introduction to permeases and other transport facilitating proteins.
A xe2x80x9ctransmitterxe2x80x9d is a transportable molecule which can be transported by the transporter, and/or which can trigger a detectable change on a cell comprising a receptor for the transmitter. Examples include neurotransmitters which transmit a signal across a synaptic junction by binding to a receptor on a post-synaptic cell, where the neurotransmitters are also transported by neurotransmitter transporters.
A xe2x80x9clongitudinal segmentxe2x80x9d includes a segment of a channel (e.g., a microchannel) that extends over at least a substantial portion of the length of the channel.
A xe2x80x9cdouble Yxe2x80x9d array is a configuration of channels (e.g., microchannels) in which at least four channels intersect with a first channel, i.e., at least two channels intersect with one end of the first channel and at least two channels intersect with the other end of the first channel.